1. Field of the Invention
This invention relates to a dry-type multilayer analytical element for analysis of an analyte (the object substance to be analyzed) in a liquid sample where the analyte directly or indirectly produces hydrogen peroxide in the presence of oxidase. More particularly, this invention relates to a dry-type multilayer analytical element for analysis of an analyte in a liquid sample containing oxidase, a hydrogen peroxide-decomposing enzyme (H.sub.2 O.sub.2 -decomposing enzyme), a ferrocyanide and a chromogen as essential components wherein the H.sub.2 O.sub.2 -decomposing enzyme is incorporated in a layer more distant from the support of the analytical element than the layer containing the chromogen.
2. Description of Prior Art
In clinical chemical analysis, a widely used colorimetric method determines the H.sub.2 O.sub.2 produced from an analyte in a sample, such as, serum by using an oxidase of which the substrate is the analyte or an oxidase of which the substrate is a substance derived from the analyte. The H.sub.2 O.sub.2 is usually allowed to oxidize a chromogen in the presence of a H.sub.2 O.sub.2 -decomposing enzyme such as peroxidase (POD). The chromogen can be divided into two groups, i.e. leuco dye type chromogens oxidized to form color materials from themselves and coupling type chromogens which are oxidized and then coupled with a coexisting coupler to form colored materials. The oxidized coupling type chromogen itself does not have a color.
Various reagent compositions have been developed for determination of analytes utilizing H.sub.2 O.sub.2 produced from them, and various analytical elements for dry-type analysis are also known. As the examples of the reagent compositions containing ferrocyanide as an essential component for determination of H.sub.2 O.sub.2, U.S. Pat. No. 3,886,045 (U.S. Re. No. 29,498) discloses a reagent composition for determination of glucose comprising glucose oxidase, POD, 4-aminoantipyrine (4-AA), a coupler, such as, phenol and ferrocyanide. U.S. Pat. No. 4,291,121 discloses a reagent composition for determination of uric acid or cholesterol comprising uricase (uric acid oxidase) or cholesterol oxidase, POD, 4-AA, a coupler, such as, 3,5-dichloro-2-hydroxybenzene sulfonic acid or its Na or K salt and ferrocyanide.
It is disclosed in these patent documents that ferrocyanide can eliminate the influence of a reductive substance particularly bilirubin in a sample which is an interfering material for the determination. However, the inventors have found that only a limited selection of chromogens must be used, because all the components are allowed to coexist irrespective of the usual chemical analysis or dry-type analytical element. Furthermore, the applicable analytes are restricted to the above three substances, and the color produced is unstable in the case of a dry-type analytical element.
Japanese Patent KOKOKU No. 37-5796 discloses another example of a reagent composition for the determination of glucose comprising glucose oxidase, POD, leuco dye and potassium ferrocyanide. All of these components are allowed to coexist. In this case, potassium ferrocyanide is added as a reductant for decoloration of the dye formed by oxidation of H.sub.2 O.sub.2 -POD.
As described above, when an analyte was determined by using a reagent composition comprising an oxidase specifically reacting with the analyte to produce H.sub.2 O.sub.2, POD, chromogen and ferrocyanide, these all components were added to one solution or one layer. As a result, coloration and decoloration proceeds in parallel, and thereby the accuracy of the results are lowered. Particularly, in the case that the evolution of H.sub.2 O.sub.2 is abundant or in the case that the chromogen employed is 3-substituted imidazole leuco dye, this phenomenon is pronounced. This matter suggest that coexisting H.sub.2 O.sub.2 is a cause of the decoloration of the color material produced.
The reaction formula in the case of cholesterol is shown below. ##STR1##
This reaction formula is suggested in Japanese Patent KOKAI No. 49-50991 and by Fossati et al. ("Clinical Chemistry", 26 (2), 227-231 (1980)). Besides, a dry-type analytical element containing these four components is disclosed in Japanese Patent KOKAI No. 56-15582. This analytical element is formed of a test paper (reagent strip) impregnated with a solution containing a reagent composition for determination of cholesterol or uric acid comprising the above four components and then dried.
A dry-type multilayer analytical element is superior to such a test paper in operation and quantitation (Asaji Kondo "Bunseki (Analysis)" 1984 (7), 534). The present inventors have prepared a multilayer analytical element for determination of cholesterol containing the above four components in one layer according to the method of D. M. Dappen et al. ("Clinical Chemistry", 28 (5), 1159 (1982)). And, they have found that in the case of this analytical element, the colored material formed was unstable, and it was decolored during measuring.
While, Japanese Patent KOKAI No. 61-124393 discloses a multilayer analytical element for detecting hydrogen peroxide containing peroxidase, a hydrogen donor corresponding to the coupling type chromogen in the analytical element of the invention and a coupler. In this patent, the separation of peroxidase from the hydrogen donor is proposed for the purpose to improve the stability in storage of the analytical element by preventing the deterioration of peroxidase. However, this patent does not refer to the incorporation of a ferrocyanide which also brings deterioration of the analytical element by reacting with other components of the reagent composition and a polymer binder.